Đề tài Types of PCR and their applications

BỘ GIÁO DỤC VA ĐÀO TẠO TRƯỜNG ĐẠI HỌC NÔNG LÂM THÀNH PHỐ HỒ CHÍ MINH BỘ MÔN CÔNG NGHỆ SINH HỌC Group : Nguyeãn Traàn Laâm Thanh Huyønh Thanh Khoa Buøi Thò Hoàng Gaám Traàn Nam Trung Ñinh Caùt Ñieàm RT - PCR ( Reverse transcriptase PCR ) In situ PCR Competitive RT - PCR Real - Time PCR PCR - ELISA (Enzyme linked immunoassay ) Rep - PCR TAIL - PCR ( Thermal asymmetric interlaced - PCR) Multiplex -PCR PCR - SSCP Inverse - PCR Alu - PCR TAIL – PCR (Thermal asymmetric interlaced-PCR) Introduction A simple and powerful tool for the recovery of DNA fragments adjacent to known sequences Was developed by Liu and Whittier in 1995 Utilizes a set of nested sequence-specific primers together with a shorter arbitrary degenerate (AD) primer The relative amplification efficiencies of specific and nonspecific products can be thermally controlled Advantages Simplicity neither special DNA manipulations before PCR (restriction digestion, ligation, etc) nor laborious screening afterward (Southern hybridization, primer labelling and extension, gel excision, etc) simple agarose gel analysis can confirm product specificity the requirement for the template DNA quantity (~ng) and purity are extremely modest High specificity the proportion of coamplified nonspecific products is very low High efficiency 60-80% of reactions yielded specific products with any given AD primer Speed The successive amplification reactions can all be completed in 1 day Less risks in chimeric artifacts TAIL PCR doesn't involve ligation step Direct sequencing The high specific reaction products can be added directly to the sequencing reaction , no gel excision and purification are required High sensitivity Single-copy sequences in genome can be amplified Principle of TAIL-PCR Important features of TAIL-PCR Primer Design Specific primer (SP) Nested sequence specific primer complementary to vector sequence High melting temperature, Tm=58-63oC Arbitrary degenerate (AD) primer Relatively shorter Lower melting temperature, Tm =47-48oC Annealing Temperature High-stringency cycle (thermal asymmetric) Annealing temperature = 63oC Reduced-stringency cycle (thermal symmetric) Annealing temperature = 44oC Low-stringency cycle Annealing temperature = 30oC Protocol of TAIL-PCR SP2 SP3 vector insert Primary PCR with SP1 and AD 5 high stringency cycles 1 low stringency cycle nontarget sequence AD primer 10 reduced stringency cycles 2 high stringency cycles (thermal asymmetric) 1 reduced stringency cycle (thermal symmetric) Nonspecific product (type II) Specific product (type I) Nonspecific product (type III) Product yield: High or middle (detectable or undetectable) High (detectable) Low (undetectable) TAIL-cycling (12 super cycles) PCR Product of Primary Reaction (B) Secondary PCR with SP2 and AD (10 super cycles) 1000-fold dilution of primary PCR product Specific product Nonspecific product (type III) Product yield: High (detectable) Very low (undetectable) PCR Product of Secondary Reaction Specific product Agarose gel analysis Direct sequencing (C) Tertiary PCR with SP3 and AD (20 normal cycles) 1000-fold dilution of secondary PCR product Cycling Orders Application High efficiency to amplify insert end segments from P1, BAC and YAC clones TAIL-PCR as a powerful tool for amplifying insert end segments from P1, BAC and YAC clones The amplified products were highly specific and suitable as probes for library screening and as templates for direct sequencing The recover insert ends can also be used for chromosome walking and mapping P1 clones YAC clones BAC clones Many product bands from the primary TAIL-PCR reaction disappeared after the secondary TAIL-PCR, indicating that these were non-specific type II products Specific products were not always seen in the primary reactions due to their low concentration. However, these specific products becomes visible after the subsequent secondary reaction Direct Sequencing Because it’s high specificity, unpurified TAIL-PCR products can be directly sequenced. Unpurified products yielded clear sequencing profiles Recovery single-copy sequences from highly complex genome Amplification of single copy sequences was found technically more difficult in organisms with large genome. e. g. Inverse PCR is difficult to apply to genomes containing over 109 bp However, TAIL-PCR is very sensitive and can be applied to highly complex genomes Rapid isolation of promoter sequences The isolation of promoter and enhancer sequences is a crucial step in the study of the regulation of gene expression Flanking regions of genes, containing these elements, were conventionally isolated by screening genomic libraries using cDNA as probes, which is very time-consuming Therefore, simpler and more reliable, and preferably PCR-based methods for promoter isolation are urgently required. Unlike Inverse PCR and ligation-mediated PCR, TAIL-PCR is a simple and efficient technique for genomic walking which does not require any restriction or ligation steps Ex: Rapid isolation of promoter seq. of Pal genes from yams Aligned DNA sequences of three TAIL PCR products obtained from the 5’-flanking regions of Pal genes of yams DNA sequences of PCR products overlapped perfectly with the 5’-end sequence of the cDNA. In the region isolated, a putative TATA box and several MREs could be identified. Isolated 5’-flanking regions of Pal and Pgi genes were fused to the GUS gene, and their activity was tested by transient transformation after delivery into tobacco BY2 cells by particle bombardment. All the isolated 5’-flanking regions were shown to drive reporter gene expression. Conclusion TAIL-PCR is highly specific and efficient for amplification of DNA segments adjacent to known sequences Upon different modification, this technique could be used to handle vary tasks: Amplification of Insert Ends fragments from P1, YAC and BAC clones for chromosome walking Isolation of 5’ flanking region of genes Isolation of promoter sequences Isolation of T-DNA insert junctions for genome physical mapping, development of sequence-tagged sites (STS), and analysis of genomic sequences flanking T-DNA, transposon or ritrovirus insertions. Mycoplasma Detection by the Mycoplasma PCR ELISA Introduction The most frequent contaminants of animal cell cultures are mycoplasmas. According to recent screenings, about 35% of all cell cultures are contaminated with mycoplasmas (1). Due to their size and lack of a bacterial cell wall, mycoplasmas are able to pass through 0.2 mm filters commonly used for sterile filtration of media and media components.Mycoplasmas cannot be detected by the light microscope. Unlike ordinary bacterial contamination, mycoplasmal infection does not cause turbidity in the cell culture medium, even if growth densities of 108particles/ml are reached. Another prominent feature of mycoplasmas is their resistance to various antibiotics used for the prevention of bacterial contamination in cell culture. An array of physiological and biochemical parameters are affected by the presence of mycoplasmas in cell culture. Various methods are available for the detection of the five mycoplasma species most commonly found in mammalian cell culture (M. fermentans, M. hyorhinis, M. orale, M. arginini, A. laidlawii; see references 2,3). These methods are based on microbiological culture, DNA staining with fluorescent dyes, immunobinding, enzymatic or serological tests, ELISA, and/or PCR. Materials and Methods Buffers and reagents of the Boehringer Mannheim Mycoplasma PCR ELISA were used as supplied with the kit. Sample 1 preparation Mycoplasmas were grown, and DNA was isolated essentially The method is derived from a “nested PCR” detection method that has been previously established (4). Sample 2 preparation Supernatants from animal cells were centrifuged for 10 minutes at 13,000 x g and4oC. Supernatants were removed carefully, and 10 ml sterile water and 10 ml of lysisreagent were added to the pellet, which was invisible in some cases. After a 1 h incubation at 37oC, 30 ml of neutralization reagent was added. F E A T U R E S Amplification In a PCR tube, 10 ml of the sample was mixed with 15 ml of sterile water and 25 ml of the ready-to-use PCR master mix. After a preincubation for 5 minutes at 95oC, DNA was amplified in Thermal Cyclers 480 and 9600 (Perkin Elmer) for 40 PCR cycles (strand separation, 30 sec at 94oC; annealing, 30 sec at 62oC; elongation,1 min at 72oC) followed by a final elongation for 10 minutes at 72oC. Detection Ten microliters of the sample was incubated with 40 ml of denaturation reagent for 10 min at room temperature. Hybridization reagent (450 ml), prepared as described by Boehringer Mannheim, was added, and 200 ml samples were transferred to streptavidin-coated microtiter plates, followed by a 3 h incubation at 37oC with constant agitation. After three washes with 250 ml of washing solution per well, 200 ml of peroxidase-conjugated anti-digoxigenin was added to each well and agitated at room temperature for 30 min. Following five washes with 250 ml washing buffer per well, 100 ml of substrate was incubated with each sample well for 5–20 min. Thereafter, 100 ml of stop reagent was added to each well, and the absorbance was subsequently measured at 450 nm in a microtiter plate (ELISA) reader. Results 1. For sample preparation, the mycoplasmas are enriched by centrifugation of the cell-free supernatants and then lysed by alkali treatment, and the lysate is neutralized. 2. A single round of PCR is performed, using a set of mycoplasma groupspecific primers that bind to conserved regions of the mycoplasmal 16S rDNA. 3. Mycoplasma-specific DNA is detected after denaturation of the PCR amplicon and subsequent hybridization with a specific, biotinylated capture probe. Specificity of the PCR ELISA procedure The specificity of the assay was determined by using proteinase K/SDS-purified DNAs from 20 chromosomal DNAs of different organisms. Fifteen DNAs were of mycoplasmal origin, including the five most common contaminants of animal cell culture. Five chromosomal DNAs were derived from species that are closely related (Lactobacillus casei, Clostridium perfringens), more distantly related (E. coli), or not related (Saccharomyces cerevisiae, mouse Ltk – cells) to mycoplasmas. The Mycoplasma PCR ELISA detected all Mycoplasma and Acholeplasma species (Table 1). Four out of five negative control DNAs were negative in this assay. A weak positive signal was obtained from Clostridium perfringens DNA. Two types of sensitivity assays were performed. First, defined amounts of purified DNA from five mycoplasmal DNAs were used, four of them representing predominant contaminants of cell culture (Table 2).The sensitivity varies, ranging from 1 pg (M. arginini, M. hominis) to <1 fg (M. fermentans, A.laidlawii). Since 1 fg approximates 1–3 copies of 16S rDNA, as little as 1–3 mycoplasma particles are detectable in the case of M.fermentans or A. laidlawii. Provided that suspected animal cells arepropagated in the absence of antibiotics,mycoplasma densities can easily reach 108 organisms/ml. Therefore, even a detection limit of 1000 particles/ml is sufficient for routine diagnosis of mycoplasma contamination of cell cultures. Supernatants from naturally infected cell cultures were taken for a second array of sensitivity experiments. In all cases, mycoplasmas were detected – even in dilutions of 105(Table 3). For further evaluation, the Mycoplasma PCR ELISA was compared to the microbiological cultivation method, which has served for a long time as the “gold standard” for mycoplasma detection methods. In two cases, the PCR ELISA data were confirmed by cultivation. Generally, due to the sensitivity limit, dilutions >103 of cell culture supernatants were not recommendable for the cultivation method. In one case, microbiological cultivation failed to detect M. hyorhinis, most probably since this species is not easily cultivatable. Taken together, the specificity and sensitivity of the Mycoplasma PCR ELISA are superior to that of known non-PCR mycoplasma assays. The advent of PCR technology combined with nonradioactive hybridization/detection methods facilitates the detection of mycoplasmas. The method combines the features necessary for an elegant detection method. Sample preparation is easy because extraction procedures are avoided. The set up of the PCR reaction is simple, employing a ready-to-use mix. Increased sensitivity and specificity are achieved by a capture hybridization followed by a nonradioactive detection cascade. Also, the appearance of false positives as a result of carry-over is reduced by omitting a second PCR step and gel analysis. However, despite the omission of a second PCR step,precautions should be followedto prevent PCR contaminations. In Situ PCR Amplification of Intracellular mRNA Introduction The polymerase chain reaction (PCR) is now commonly used m laboratories involved in research studies and clinical diagnostic work (I,2). A major advantage of PCR combined with reverse transcription (RT-PCR) 1s that it can be used to amplify and detect rare mRNA within a specimen. However, conventional RT-PCR cannot be used either to quantitate the frequency of cells expressing a particular mRNA or determine the cellular origin of the amplified signal. Both of these factors may be relevant in the interpretation of gene expression. In order to overcome the limitations of conventional RT-PCR, methods have been developed for performing in situ RT-PCR (for reviews, see 3-6). By performing the reverse transcription and subsequent amplification within the cells fixed onto microscope slides, it is possible to identify the cellular origin of the signal. The technique has an advantage m that it does not require mRNA to be extracted from the sample, and thus, there is no potential for signal loss during the nucleic acid tsolation step. Furthermore, unlike conventional PCR, the technique can be used to determine the prevalence of gene expression within a cell population. The technique is based on the functional hypothesis that enzymes and reagents can freely enter fixed cells, and synthesize and amplify cDNA in situ. One important corollary is that the PCR products themselves can also freely enter and egress. Consequently, the success of the technique is dependent on an equilibrium between permeability to reagents and the retention of PCR products. In our experience, we found that carefully controlled fixation and digestion were important in maximizing the in situ amplification and retention of signal. The optimization of the equilibrium is critical to the success of the techmque, and careful calibration of reaction conditions is mandatory for each primer set used. we will describe the technique we developed for the detection of granzyme A and perforin mRNA in cytospin preparations of activated human peripheral blood lymphocytes . 2. Materials 2.1. Glass Slide Preparation 1. Glass slides (Solmedia, Romford, Essex, UK). 2. Decon 90 (Decon Laboratories, Hove, UK). 3. 3-Aminopropyltriethoxysilane (Tespa; Sigma, Poole, Dorset, UK). 4. Acetone (Merck, Poole, Dorset, UK). 5. Diethyl pyrocarbonate (DEPC; Sigma) treated doubly-distilled water (DEPC- ddH2O; see Note 1) 6. Coverslips (Chance Propper, Smethwick, Warley, UK). 7. 1% Dimethyl dichlorosilane in Ccl, (Merck). 2.2. Cell Preparation 1. Anticoagulated human peripheral blood. 2. RPM1 (Imperial Laboratories, Andover, UK) supplemented with 10% fetal calf serum (FCS; Imperial Laboratories), 2 mM L-glutamme, 100 mg/mL penicillin, and 100 &nL streptomycin. 3. Lymphocyte separation medium (Flow Laboratories, Rickmansworth, He&., UK). 4. Phosphate-buffered saline (PBS; Unipath, Basingstoke, UK). 5. Phorbol-12-myristate-13-acetate (PMA, 500 ng/mL; Sigma). 6. Phytohemagglutinin (PHA, 1 mg/mL; Wellcome, Dartford, UK). 7. Tissue-culture incubator at 37oC. 8. Cytospin centrifuge (Shandon Southern Products, Runcom, Cheshire, UK). 2.3. Fixation Starting from this step, it is imperative that all glassware is baked and all reagents are RNase-free (see Note 2). Chemicals should be reserved for RNA work. 1. 4% Paraformaldehyde (Sigma) in DEPC-PBS: Make fresh; heat suspension to 60oC with constant agitation for at least 1 h to allow paraformaldehyde to dissolve in DEPC-PBS. Cool to room temperature before use. 2. DEPC-3X PBS, DEPC-1X PBS. 3. DEPC-ddH2O. 4. 50% Ethanol in DEPC-ddHZO, 80% ethanol in DEPC-ddH20, 100% ethanol. 2.4. Proteinase K Digestion Stock solutions: 1. Proteinase K (Type XI protease [Sigma] 10 mg/mL in 0. IM Tris-HCl, pH 8.0). 2. 1M Tris-HCl, pH 8.0. 3. 500 WEDTA, pH 8.0. 2.5. Hybridization and Reverse Transcription 1. Hybridization solution: 50% formamide (Merck), 10% dextran sulfate (Pharmacia, Milton Keynes, Bucks., UK), 300 mM NaCl, 20 mM Tris-HCl, pH 7.6, 5 mM ethylenediaminetetraacetic acid (EDTA), 1X Denhardt’s (Sigma), 10 mM dithiothreitol (DTT, Sigma). 2. Antisense primer at 1 mg/mL (see Section 2.6.). 3. Moloney murine leukemia virus reverse transcriptase (RT; Life Technologies, Uxbridge, UK). 4. 5X Reverse transcription buffer (Life Technologies). 5. 100 mM stocks of dATP, dCTP, dGTP, dTTP (Boehringer Mannheim, Lewes, East Sussex, UK). 6. 100 mMDTT (Life Technologies). 7. RNase inhibitor (Promega, Southampton, UK). 8. Bovine serum albumin (BSA [Sigma]) 10 mg/mL in DEPC-H20. 9. DEPC-ddH20. 10. DEPC-2X SSC. 11. Humidified slide box. 12. Hybridization oven at 42OC. 13. Hybridization oven at 37oC. 2.6. In Situ PCR 1. Granzyme A primers: 5’-CCA GAA TCT CCA TTG CAC GA 5’-CTG TAA CTT GAA CAA AAG GT 2. Perforin primers: 5’-ACA TGG AAA CTG TAG AAG CG 5’-GGA TTC CAG CTC CAT GGC AG 3. Taq polymerase (Promega). 4. 1 OX Taq polymerase buffer (Promega). 5. 100 mM stocks of dATP, dCTP, dGTP, dTTP. 6. 22.5 nmol biotin-1 I-dUTP (Sigma). 7. 25 mM MgCl, (Life Technologies). 8. DEPC-ddH2O. 9. Mineral oil (Sigma). 10. Xylene (Merck). 11 Thermocycler (Hybaid, Teddmgton, Middlesex, UK). 2.7. Detection of Amplified Products 1. Mouse antibiotin monoclonal antibody (MAb) (Dako, High Wycombe, Bucks., UK). 2. Horseradish peroxldase (HRP)-conjugated, rabbit antimouse Ig antibody (Dako). 3. Human AB serum. 4. BSA (10% stock solution m PBS). 5. Substrate: 3,3-diaminobenzidine tetrahydrochloride (DAB [Sigma], 0.6 mg/mL in PBS made freshly with the addition of 3 mL/mL 3% H202 (Thornton and Ross, Huddersfield, UK) immediately before use. 6. Harris’ hematoxylin (Sigma). 7. 70 ,90, and 100% Ethanol. 8. DPX mountant (Merck). 9. PAP pen (Bayer Diagnostics, Basingstoke, Hams., UK). 10. Humidtfied staining tray. 3. Methods 3.1. Glass Slide Preparation 1. Glass slides should be thoroughly cleaned by soaking overnight m 10% Decon 90 in double distilled water. Then rinse slides successively with coprous amounts of hot tap water, deiomzed water, and ddH20, place in racks, dry, wrap in alummum foil, and bake at 200oC for 4 h to destroy RNase activity. From this point onward, all glassware used should be RNase free (see Note 1). Then coat slides by incubating m a solution of 2% Tespa in acetone for 2 mm. Rinse twice with fresh acetone, twice with DEPC-H20 (see Note l), wrap loosely in aluminum foil, and dry at 37OC overnight. Slides may be stored at room temperature before use. 2. Coverslips should be silicon-coated for easy removal during the procedure. Soak the coverslips in 1% dimethyl dichlorosilane in Ccl4 for 1 min, rinse with fresh ddH2O, wrap in alummum foil, bake at 200oC for 4 h, and store at room tempera- ture until required, 3.2. Cell Preparation 1. Dilute human peripheral blood 1:2 with PBS and isolate lymphocytes by cen- trtfugation through lymphocyte-separation medium at 400g at 20oC for 25 min. 2. Collect lymphocytes from the gradrent interface and dilute at least 1:2 in PBS; pellet at 400g at 20oC for 7 min. 3. Wash twice further in PBS, centrifuging at 300g at 20oC for 5 min. 4. Resuspend cells in supplemented RPM1 medium at a concentration of 1 x 106 cells/ml, and stimulate with 100 ng/mL PMA and 50 mg/mL PHA in RPMI/FCS. 5. Four days later, harvest cells by centrifugation at 300g at 20oC for 5 min. 6. Wash cells m DEPC-PBS buffer and pellet onto Tespa-treated microscope slides by Cytospin centrifugatron at 80g at 20oC for 5 min. 3.3. Fixation 1. Place Cytospin preparations on Tespa-treated glass slides in baked slide racks. 2. After allowing the slides to dry for 5 min, fix the cytospin preparations at room temperature according to the following schedule (see Note 2): a. 4% pamformaldehyde, 20 min; b. 3X DEPC-PBS, 5 min; c. 1X DEPC-PBS, 5 min; d. 1X DEPC-PBS, 5 min; e. DEPC-ddH,O, 1 min; 50% EtOH in DEPC-ddH2O, 1mm; f. 80% EtOH in DEPC-ddIH2O, 1 min; and g. 100% EtOH, 1 min. 3. After fixation, cytospm preparations may be covered in aluminum foil and stored at -80oC. 3.4. Proteinase K Digestion 1. Place slides in a baked 2-L beaker at 37oC for a 30-min digestion with 10 mg/mL proteinase K in 0.1M Tris 0.5 mM EDTA, pH 8.0 (see Note 2). 2. Subsequently fix slides in 4% paraformaldehyde as in Section 3.3. 3.5. Hybridization and Reverse Transcription For all of the manipulations described below, the slides are placed on a bench covered with aluminum foil. 1. Hybridize cytospin preparations with 10 mL of hybridization solution containing 2.5 ng/mL of antisense oligonucleotides to the human granzyme A or perform genes in a humidified chamber for 2 h at 42OC (see Note 3). 2. During this incubation, the preparations are covered with a coverslip, and carefully placed onto the cells using baked forceps. 3. At the end of the incubation, wash slides vigorously in 2X SSC for 5 min to remove coverslips and hybridization buffer. 4. Shake slides vigorously, wipe with tissue to remove excess salt, and then air-dry. 5. Apply 7 & of reverse transcription mixture to the cell pellet, cover with a cover- slip, and incubate for 1 h in a humidified chamber at 37oC. 6. The mixture contains reverse transcriptase (3 U/mL), in a buffer of 75 mM KCl, 10 mMTris, pH 8.0, 12 mMMgC2, 2 mg/mL BSA, 10 mMDTT containing 1 mM of each dATP, dGTP, dCTP, dTTP, and 1 U/mL RNase inhibitor. 7. Wash slides extensively in 2X SSC buffer for 5 min, briefly rinse in ddH2O, and nair - dry. 3.6. In Situ PCR Controls are critically important for in situ PCR. (Please refer to Notes 4 and 5 for suggestions.) In order to reduce the quantity of reagents required for the PCR stage, cut coverslips to a size of approx 1 cm* using a diamond glass cutter. 1. Add 5mL of a solution containing Tug polymerase at 0.5 U/mL; 1 mM dATP, dGTP, dCTP; 0.9 mMdTTP; 0.1 mM biotin- 11 -dUTP; 75 mM KCI; 10 mM Tris, pH 8.0; 10 mM MgCl2; and 7 pmol/mp of each 5’- and 3’-oligonucleotide complementary either to the human granzyme A or perforin genes to the slides (see Note 6). 2. Place slides on the thermocycler (see Note 7), cover the mixture with the small coverslips, and flood the slides with mineral oil to prevent desrccatron (see Note 8). 3. The amplification proceeds for one cycle at 94OC for 5 min; 30 cycles at 94oC for 1 min, 60oC for 1 min, 72OC for 1 min; one cycle of 72OC for 10 min 4. After PCR amplificatron, submerge the slides in xylene for 2 min to remove min- eral oil and then leave in the fume hood to allow xylene to evaporate. 5. Spray the slides with 70% alcohol and wipe with tissue to remove any remainmg oil (see Note 9). 6. When dry, place the slides m a rack and agitate in 2X SSC to remove coverslips. Then wash extensively in 2X SSC, in PBS, and finally air-dry. 3.7. Defection of Amplification Products 1. Draw a circle around the cell pellet with a PAP pen in order to create a barrier to contain reagents for the detection step. 2. The detection step IS performed in a humidified chamber. 3. Rinse slides in PBS and incubate for 30 min with 50 mL of mouse antibiotin MAb (1:25 in PBS containing 0.5% BSA). 4. Wash the slides three times in PBS, incubating for 5 min each time. 5. Detect the primary antibody by incubating for 30 min with 50 mL of HRP-coqu- gated rabbit anttmouse Ig antibody ( 1:50 in PBS with 10% human AB serum and 0.5% BSA). 6. After washing a further three times in PBS, develop the signal with the freshlyb prepared substrate solution (DAB and H2O2). 7. Counterstam with Harris’ hematoxylin. 8. Dehydrate through 70,90, and 100% ethanol (1 min each), equilibrate in xylene, and mount in DPX medium. Figure 1 illustrates results obtained by using the RT-PCR technique described in this chapter showing granzyme A mRNA in stimulated peripheral blood lymphocytes. 4. Notes 1. It is important to use RNase-free conditions for this technique. Solutions should be treated with 0.1% DEPC for 12 h at 37oC and autoclaved for 30 min before Fig. 1. In situ cDNA PCR detection of granzyme A. (A) Granzyme A was detected in PHA/PMA stimulated lymphocytes following 30 cycles of PCR amplification. (B) PHA/PMA-stimulated lymphocytes were mixed 1:4 with unstimulated, granzyme A-negative, peripheral blood lymphocytes. Large blast cells are granzyme A-positive, whereas the smaller, unstimulated lymphocytes are negative. This demonstrates that,in this system, despite the presence of labeled PCR product in the supernatant, cells negative for granzyme A within a mixed-cell population remain unstained. Use. Tris buffers cannot be treated directly with DEPC, but should be made with DEPC-treated, autoclaved ddH2O. Glassware should be rendered RNase-free by covering with aluminum foil and baking for 4 h at 200oC. Gloves should be worn at all times. When it is necessary to place slides on the bench, the bench should be covered with aluminum foil and the slides manipulated with baked forceps. 2. Cellular fixation and digestion are critical to the success of this technique. The conditions may vary according to the type of cells used, the size of the PCR product, and the stability of the mRNA. The conditions need to be optimized for each new set of experiments. 3. The duration of hybridization needs to be empirically determined. Although a longer hybridization time should favor oligonucleotide-mRNA binding, labile mRNA may degrade, resulting in a truncated cDNA. 4. Controls are of crucial importance in in situ PCR systems. Positive controls using primers specific for a housekeeping gene need to be performed on the test cells to demonstrate the presence of mRNA. Cells known to be positive for the gene of interest should be included to demonstrate that the reaction conditions have been optimized. Negative controls are particularly important in a system such as we describe, where labeled nucleotides are directly incorporated into the PCR prod- uct. In this respect, a PCR in situ hybridization system has advantages and may be preferred. Some suggested negative control reactions are shown in Table 1. A negative result from reaction (a) will demonstrate the absence of endogenous peroxidase in a sample and from (b) the absence of nonspecific binding of the HRP-conjugated secondary antibody. Reactions (c) and (d) will show that there is neither endogenous biotin in the sample nor a signal following RT alone. Table 1 Suggested Negative Controls for In Situ cDNA PCR Reactions (e) and (f) are particularly important controls; a negative result demonstrates that the signal results from amplification of the cDNA, and not from priming by nicked DNA or amplification of genomic DNA. Reaction (g) demonstrates the requirement for primers and (h) on Taq polymerase to produce a signal. 5. It 1s possible to perform a Southern blot analysis on the supernatant from the zn situ PCR reaction (3). This enables the specificity of the amplified product to be confirmed. 6. In srtu PCR appears to require higher concentrations of reagents than tube PCR (MgCl2, Taq polymerase, and nucleotides). Consequently, it may not be possible to transfer conditions optimized for tube PCR directly to an in sztu PCR system. 7. The heating blocks of traditional PCR thermocyclers, with their discontinuous surface area, do not provide ideal heat conduction for in sztu PCR. It is preferable to use a machine with specifically designed flat blocks. 8. Desiccation during the PCR process can yield false-positive signals. It is impor-tant that the mineral oil completely seal the coverslip. 9. The mineral oil should be completely removed before the immunohistochemistry step. If present, the hydrophobic oil droplets will interfere with the antibody binding and subsequent detection of the PCR product. Multiplex PCR design strategy used for the simultaneous of 10 Y chromosome short tandem repeat (STR) loci Abstract The simultaneous amplification of multiple regions of a DNA template is routinely performed using the polymerase chain reaction (PCR) in a process termed multiplex PCR. A useful strategy involving the design, testing, and optimization of multiplex PCR primer mixtures will be presented. Other multiplex design protocols have focused on the testing and optimization of primers, or the use of chimeric primers. The design of primers, through the close examination of predicted DNA oligomer melting temperatures (Tm) and primer–dimer interactions, can reduce the amount of testing and optimization required to obtain a well-balanced set of amplicons. The testing and optimization of the multiplex PCR primer mixtureconstructed here revolves around varying the primer concentrations rather than testing multiple primer combinations. By solely adjusting primer concentrations, a wellbalanced set of amplicons should result if the primers were designed properly. As a model system to illustrate this multiplex design protocol, a 10-loci multiplex (10plex) Y chromosome short tandem repeat (STR) assay is used. Introduction Multiplex polymerase chain reaction (PCR) is defined as the simultaneous amplification of multiple regions of DNA templates by adding more than one primer pair to the amplification reaction mixture. Described in1988. Materials and methods Primer design and synthesis Sequence searches and alignments PCR reaction and thermal cycling conditions Capillary electrophoresis of PCR products Table 1 Y STR loci used in the Y STR 10plexa Size ranges calculated using the GenBank sequence as the reference allele. The GenBank accession numbers given are BAC clones containing STR sequences. Size ranges given are based on sequence information given in GenBank, and take into account the adenylation of PCR products through non-template addition. All primers were redesigned except DYS19 and DYS437 in order to fit into the multiplex design. Multiplex PCR primer design Select loci to include in multiplex Define reference sequence using GenBank Sequence alignments Fig. 2 Sequence alignment of the Y STR marker GATA A7.1 from GenBank accession numbers AC009235 (10 GATA repeats) and G42675 (11 GATA repeats). The alignment of sequences from multiple individuals is useful to search for possible polymorphic nucleotides that might impact primer annealing and subsequent amplification. For example, an extra “T” was observed in G42675 (see the boxed region). The dotted line arrows represent the primers from the original reference describingthis marker [31]. The solid line arrows represent the primer positions described in this work. The boxed areas represent differences in sequence between the two accession numbers. The alignments were performed using the BCM Search Launcher: Multiple Sequence Alignments program located at the following URL: Allele and size range determination and multiplex schematic Fig. 3 Schematic of size ranges and dye label colors used in designing this Y STR 10plex assay. The length of the box containing the locus name represents the size ranges(listed above box) for the known alleles (listed below box). Three different fluorescent dyes are used in this Y STR 10plex that are spectrally distinguishable using Filter C on the ABI 310: 6 -FAM(blue), TET (green), and HEX(yellow). Primers in this multiplex were redesigned compared to previous studies except those used to amplif DYS19 and DYS437 (see Table 1) Primer design Table 2 Primer sequences used in this studya F refers to the forward primer and R refers to the reverse primer for a particular locus. FAM, TET, and HEX are commercially available fluorescent dyes Predicted primer melting temperatures (Tm) were calculated using a total primer concentration of 0.05 µM and [Na+ ]= 50 mM and equations from Rozen and Skaletsky [24] Check and compare Tm Primer–primer comparison Table 3 Crosschecking performed on the Y STR 10plex a primers. Potential primer pairinteractions are represented Level of interaction based on an alignment score which is defined as the number of complementary base pairs minus the number of mismatched base pairs between two primers. BLAST search for newly designed primers Primer modification (“G” tail and fluorescent dye labels) A Multiplex design, starting with the selection of loci to be included in the multiplex and ending with the purchasing of the desired primers. Multiplex PCR optimization and testing Primer quality Fig. 4 Comparison of ABI 310 electropherograms from singleplex PCR reactions of the Y STR GATA H4 marker amplified with dye-labeled primers that were purified differently. The reversedphase HPLC purified NED-labeled primer (top panel) produced a clean signal with a nice flat baseline. The affinity matrix purified TAMRA-labeled primer (bottom panel) showed multiple dye blobs in the region of 75–425 bp. These dye impurities failed to be removed after primer synthesis with the affinity matrix purification procedure and can interfere with detection of true alleles present from other locus amplified in a multiplex reaction. PCR product was generated as described in the materials and methods section. Test primers in singleplex PCR Multiplex PCR testing Balance mix empirically based on PCR product yields Fig. 5 Comparison of ABI electropherograms of PCR amplicons generated with the Y STR 10plex primer set listed in Table 2. The top two panels show the result from two different male DNA samples while the bottom panel is from a female DNA sample. Failure to detect PCR products from the female sample demonstrates that the primers in the Y STR 10plex are male-specific. PCR and electrophoresis conditions as described in materials and methods B Multiplex optimization and testing, starting with a check of the quality of each primer and ending with balancing the primer concentrations based upon PCR product yields. Discussion Multiplex PCR primer selection is a complex process that can be stratified into logical steps to obtain a working primer mix. Successfully optimized multiplex PCR primer pairs should be able to amplify of all desired loci, achieve similar yields between respective amplicons, be absent of non-specific PCR amplicons, and have all amplicons labeled with a particular fluorescent dye resolvable no matter what the size of alleles present. Additionally, the introduction of this Y STR 10plx could prove valuable to the forensic community. It provides scientists with a working multiplex assay that is male specific and gives results on 10 Y STR markers simultaneously. Increasing the number of loci simultaneously amplified is restricted by several factors. The first is size. Amplicons should be designed that are at least 75 base pairs in size. The most common algorithm used for determining the DNA fragment size is known as the local southern method, and works very well for accurate sizing of DNA fragments over the 100–450 bp size range [25]. The local southern sizing method uses two sizing peaks below and two above the amplicon to determine its size. In CE electropherograms, the 35 bp and 50 bp can sometimes not be sized. This happens because these sizing fragments are often concealed by the peaks caused by unincorporated primers. Thus, sizing at below 75 bp can be inaccurate. For this reason amplicons below 75 base pairs should be avoided if one intends to use CE and the local southern method as the primary means of sizing analysis. Larger STR alleles (>400 bp) are not desirable because of allelic dropout of larger alleles in STR markers caused by preferential amplification of the smaller alleles in the multiplex, or if highly degraded DNA samples are being examined [38, 39, 40, 46]. Second, the more polymorphic a particular locus is the larger the size range it will encompass. One may have to decide between two less polymorphic markers or one with a larger potential size range of alleles. Third, the lack of adequate instrumentation may curtail the amount of amplicons that can be simultaneously detected. Multicolor fluorescence detection is necessary to resolve similar sized PCR products that are labeled with spectrally distinguishable dyes. Instruments, such as the ABI Prism 3100 Genetic Analyzer 16-capillary array system, with a five dye capability can expand the analysis of multiple PCR amplicons. Amplification products of similar size can be distinguished by using different fluorescent dye labels for each one. Based upon these factors, a multiplex containing 40 different primer–pairs is theoretically possible if PCR product size ranges are extended to approximately 500 bp and STR loci are selected with moderate allele ranges. With the exception of an in-house computer algorithm used to check for primer dimers, the multiplex design approach presented here uses publicly available software tools like GenBank, BLAST, BCM sequence alignment, and Primer3. This upfront utilization of publicly available informatics can reduce the labor-intensive empirical studies common to multiplex PCR optimization. An effective and working multiplex PCR primer mixture can be constructed quickly. This multiplex design strategy is currently being used to expand the Y STR multiplex to simultaneously amplify 20 or more loci in a single reaction (23). V. PCR-SSCP Analysis of Polymorphism. 1. Introduction: - Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) is a simple method that allows one to rapidly determine whether there are sequence differences between relatively short stretches of DNA. Coupled with sequence analysis, SSCP is an extremely useful method for both identifying and characterizing genetic polymorphisms and mutations. - SSCP has been widely used to identify mutations in host genes and in viruses, such as simian immuno-deficiency virus (SIV), during the course of infection. SSCP has been used to identify and characterize polymorphisms in a variety of genes and was effective in characterizing alleles of linked genes present in individual sperm. - Orita and colleagues developed the SSCP method in 1989 and since then, it has been applied to screen for sequence differences in either genomic or complementary DNA (cDNA) samples. 2. Materials. Thermal cycling (PCR) machine. AmpliTaq® DNA polymerase, 5 U/µL (stored at –20°C). 10x PCR buffer stored at –20°C): 100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2. dNTP (1.25 mM) stored at –20°C. Primers diluted to a concentration of 20 µM . [32P]-dCTP (New England Nuclear, Boston, MA, BLU013H, 3000 Ci/mmol). DNA templates (100 ng/pL genomic or 1: 10 diluted cDNA). 0.1% SDS and 10 mM EDTA, pH 8.0. Loading buffer: 10 mM NaOH, 95% formamide, 0.05% bromphenol blue, 0.05% xylene cyanol. Long RangerTM 50% stock gel solution (FMC BioProducts, Rockland, ME). 10x TBE buffer: 0.89 M Tris-borate; 0.02 M EDTA, pH 8.0. TEMED. 10% Ammonium Persulphate (APS, after dissolving in water, store at –20°C). Glycerol (Ultra Pure, Life Technologies, Gaithersburg, MD). Sigmacote (Sigma Chemical Co., St. Louis, MO). Acrylamide gel electrophoresis supplies and equipment . Whatman 3MM chromatography paper. Plastic wrap (such as Saran Wrap). Vacuum gel dryer. Gel cassette with an enhancing screen. X-ray films. 3. Methods. a. PCR Amplification - The PCR should contain the following ingredients (in µL): 10x PCR Buffer (2.0); 1.25 mM dNTP (3.2); 20 µM 5′ primer (1.0); 20 µM 3′ primer (1.0); 5 U/µL AmpliTaq® (0.1); Experimental template (2.0); [32P]-dCTP (0.1); and H2O (10.6) for a total 20µL. - PCR amplification is performed for 30 cycles (denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 90 s) with a final extension at 72°C for 7 min. PCR amplified DNA may be kept at 4°C or stored at –20°C. b. .SSCP Gel Preparation - Prepare a 6% nondenaturing polyacrylamide gel with 10% glycerol as following: Long Ranger gel solution (7.5 mL), glycerol (7.5 mL), 10x TBE (4.5 mL), H2O (55.7 mL); 10% APS (0.4 mL), and TEMED (0.04 mL) for a total of 73.64 mL. Add TEMED and APS right before pouring the gel. - Place a few drops of Sigmacote on the inner side of the shorter plate and spread evenly with a paper towel. Allow to air dry. Sigmocote forms a tight microscopically thin film of silicone on glass, which prevents the SSCP gel from sticking to this particular plate when you separate the two plates after the gel run is done, allowing the gel to remain smooth on the other plate. Set up the plates with 0.4-mm spacers on the sides and with a strip of Whatman chromatography paper at the bottom. Clamp three sides of the plates leaving the top open. - Immediately before pouring the gel, add TEMED and APS to the mixture, collect it into a 60-mL syringe, and push the solution into the plates slowly while holding the plates at an angle. Place either a 64-well or 36-well comb into the top of the gel and clamp the top of the plates. The gel will be polymerized in 2 h. c. Dilution and Denaturation of PCR Products - Dilute 5 µL of amplified DNA in 45 µL of 0.1% SDS and 10 mM EDTA. - Mix 5 µL of above diluted PCR products with 5 µL of loading buffer. The sample is ready to load and may be stored at –20°C for 2 wk d. Loading Samples and Running Gels - Before running the gel, denature the diluted PCR samples by heating at 95°C for 2 min and cool rapidly in an ice bath. - Load 2 µL (for 64-well combs) or 5µL (for 32-well combs) of the diluted PCR products onto gel. The leftover samples can be reused if they have been stored at –20°C for less than 2 wk. - Electrophoresis is performed by using 0.6x TBE buffer at a constant 30 watts (W) for 4 h at room temperature. e. Drying Gels and Developing Films - After migration, the gel is transferred onto a double layer of Whatman Chromatography paper and covered with plastic wrap avoiding air bubbles. - The gel is vacuum dried in a gel dryer at 80°C for 1 h, and then the gel is placed facing up in a film cassette. - Expose gel to an X-ray film (the X-ray film should be put in between the enhancing screen and the gel) at –80°C for 16 to 20 h . - Warm up film or air dry film before developing. Cut a corner of the film before developing. f. SSCP Gel Analysis - SSCP patterns are analyzed upon visual examination. Analysis is facilitated with knowledge of the gene sequence, which is often available because sequence information was necessary to derive primers. Inclusion of control samples of known sequence allow allele typing.